Endurance exercise and high altitude are two common ways to improve the exercise performance by adaptations in skeletal muscular cells. The hypothesis that the adaption of muscle metabolism due to exercise training is caused by cellular hypoxia in the muscle tissue will be discussed by comparing the effects of high altitude and exercise training on skeletal muscle. Endurance exercise improves the oxidative capacity in the muscle cells by increased mitochondrial density, increased mitochondrial enzyme activity and a more efficient energy coupling and indicates an adaptation of the energy metabolism. The mitochondrial DNA is regulated by TFAM, which expression is induced by P53. HIF-1 increases the expression of PDK-1, which inhibits the pyruvate dehydrogenase complex (PDHc). The inhibition of PDHc leads to reduced mitochondrial function, what results in an decreased oxygen consumption. High altitude reduces the partial oxygen pressure in blood levels, what causes reduced haemoglobin-oxygen saturation and reduced total oxygen content of blood. To compensate for hypoxic stress, the oxidative phosphorylation reduces and the anaerobe fermentation increases. The glycolytic pathway increases to maintain the cellular ATP levels. This switch is regulated by HIF-1. With this switch, the oxygen consumption reduces. If the mitochondrial density changes remains controversial, although it is clear that the relative subsarcolemmal mitochondrial density is reduced. High altitude and endurance exercise have some effects in common and it is probably is the induced change of energy metabolism caused by a common pathway.
Athletes are looking for the best form of training to improve their performance. There are two common ways to improve aerobic performance: endurance exercise training and staying at high altitude. Both challenges have shown to improve endurance exercise performance, which is generally viewed as the body’s adaptations to environmental alterations to maintain homeostasis (Hoppeler & Vogt, 2001).
Exercise training is known to induce adaptations in the cardiovascular system as well as in skeletal muscles. This thesis focuses on the adaptations of the skeletal muscle tissue to exercise training. These muscular adaptations, which will be mentioned later on, might be a response to cellular hypoxia, a condition of insufficient oxygen in the cells for the -aerobic energy metabolism. For exercise causes increased oxygen consumption, which results in reduced oxygen levels in the exercising skeletal muscles. (Lindholm & Rundqvist, 2016)
Environmental hypoxia at altitude is known as a key environmental stressor that initiate important physiological adaptations in the athlete. Various physiological adaptations that occur after a prolonged (> 28 days) stay at high altitude may be exploited to yield significant improvements in sea-level athletic performance (Flaherty, O’Connor, & Johnston, 2016). An important adaptation to altitude exposure is an improved oxygen carrying capacity of the blood. In response to hypoxia, the kidneys produce erythropoietin (EPO). EPO promotes erythropoiesis in the bone marrow and thereby improves oxygen carrying capacity in the blood. The production of EPO is promoted by Hypoxia-Inducible Factor-1 (HIF-1), which protein levels are increased in skeletal muscle during hypoxia for cellular hypoxia prevent the catabolism of HIF-1α (Flaherty et al., 2016).
In hypoxic cells is hypoxia inducible factor 1 (HIF-1) present, while in non-hypoxic cells HIF-1 was not detectable (Semenza, 1999). HIF-1α is a subunit of HIF-1 and regulated by cellular oxygen levels: under normoxia HIF-1α is hydroxylated and degraded, while under hypoxia HIF-1α is stable and translocated to the nucleus. That is why under hypoxic conditions higher concentrations of HIF-1 are found. HIF-1 appears to play a critical role in cellular and systemic O2-homeostasis during both development and postnatal life. Different genes with an hypoxia response element (HRE) seemed to have a higher gene transcription in cells which were incubated under 1% O2 relative to expression at 20% O2. (Semenza, 1999)
HIF plays a role in the switch from oxidative to glycolytic metabolism. This is achieved by the induction of the expression of (1) glucose transport and glycolytic enzymes, which increases the flux from glucose to pyruvate. (2) PDK-1, which blocks the conversion form pyruvate to Acetyl CoA and (3) lactate dehydrogenase A, which converts pyruvate to lactate. (Kim, Tchernyshyov, Semenza, & Dang, 2006). HIF-1 turns on different transcription genes including genes responsible for glycolysis, glucose uptake, angiogenesis and the erythropoietin (EPO) gene. Reduced skeletal muscle HIF-1 seems to improve the oxidative capacity and endurance performance (Lindholm & Rundqvist, 2016). For HIF-1 induces (indirect) glycolysis, glucose uptake and angiogenesis, HIF-1 decreases the oxygen consumption and increases the oxygen delivery to cells.
Endurance exercise and prolonged stay at high altitude have two things in common: first of all, both lead to reduced cellular oxygen levels. Secondly, they both lead to adaptations which increase the endurance performance. These corresponding effects suggest a common pathway by which endurance performance is increased. If high altitude has the same effects as endurance exercise it is plausible that their muscular adaptations are caused by cellular hypoxia.
In this thesis, the hypothesis that the adaption of muscle metabolism due to exercise training is caused by cellular hypoxia in the muscle tissue will be discussed by comparing the effects of high altitude and exercise training on skeletal muscle. In the first chapter the adaptations of muscle tissue in response to exercise training will be discussed and explained, beside that it discusses if there is any evidence that proves cellular hypoxia will occur in the muscle during exercise. The second chapter declares which mechanism may lead to adaptations to cellular hypoxia. In the third chapter the adaptations in the muscle tissue in response to high altitude will be discussed and declared. In the fourth chapter some studies that combine endurance exercise training and high altitude will be discussed. Finally, based on these findings, the reliability of the hypothesis that the muscular adaptations to exercise training are caused by cellular hypoxia in muscle tissue will be discussed.
1. Endurance exercise muscle
In this chapter the different muscular adaptations in response to endurance exercise will be discussed. We will conduct an answer to the question which muscular adaptation are known and if there is any evidence for cellular hypoxia during endurance exercise. In the first paragraph muscular adaptation to endurance exercise will be discussed and in the second paragraph we will discuss cellular hypoxia during endurance exercise.
1.1 Muscle adaptations to endurance exercise
Endurance exercise is known for aerobic improvement. In this paragraph the muscular adaptations in response to endurance exercise will be discussed. The three main adaptations are increased capillarization, increased mitochondrial enzyme activities and mitochondrial volume densities, which enhance the local capacity for oxygen utilisation (Rösler et al., 1985).
During skeletal muscle contraction reactive oxygen species (ROS) are produced, which have a regulatory role in skeletal muscle adaptation to endurance exercise (Morrison et al., 2015). Acute exercise increases oxidative stress in the skeletal muscle, what is measured by oxidized glutathione (GSSG) and F2-isoprostanes. Acute exercise increases mRNA levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and PGC related coactivator (PRC). After endurance training GPx1 mRNA expression was increased and the activity of citrate synthase and SOD enzyme was significantly increased. SOD2 protein was also increased after endurance training. (Morrison et al., 2015) Exercise training improves oxidative capacity and antioxidant capacity (Bouitbir et al., 2012). Bouitbir et al compared the oxidative capacity and antioxidant capacity in trained rats compared to the control group after exposure to atorvastatin, which is known as inducer of oxidative stress. In the trained group the maximal oxidative capacity and H2O2 production increased compared to the control group, but the free radical leak was similar in both groups. The free radical leak is a good index of mitochondrial ROS production. To test the protective effect of exercise training, rats were trained over 10 days to induce muscular adaptations. In trained rats the mitochondrial respiration was less inhibited and the maximal oxidative capacity (Vmax) was less affected by atorvastatin. These difference could be related to the generation of ROS, after the measurement of H2O2 production, the free radical leak seemed to be lower in trained muscle. (Bouitbir et al., 2012) This experiment shows that exercise training leads to inhibition of the ROS production and indicates an adaptation of the energy metabolism.
Endurance exercise training improves the oxidative capacity by increased volume density of mitochondria and improved ATP production in these mitochondria. (Camera, Smiles, & Hawley, 2016; Conley, Jubrias, Cress, & Esselman, 2013; Rösler et al., 1985) After endurance training maximal oxygen uptake (VO2max) increases. This is supported by the studies of Conley et al. and Rösler et al. Conley et al. studied the effects of endurance training in elderly. Increased VO2max, increased Pmax (power output at VO2max), an increased mitochondrial phosphorylation capacity and an increased phosphorylation capacity per unit mitochondrial volume were measured. Beside that the Pmax changed, the Pmax per unit ΔVO2max changed also significantly, that indicates an changed energy coupling. There is no evidence found for higher glycolytic ATP production or a switch to more efficient muscle fibers. The increased Pmax was associated with the rise in mitochondrial ATP production. Improved energy coupling at the mitochondrial level was evident from the rise in phosphorylation capacity per unit mitochondrial volume found in these subjects.
Rösler et al. measured an increased VO2max and maximal power output after 8 weeks of endurance training in males. The lactate levels reached 4 mM at a higher output, what means that aerobic ATP production last longer. Endurance training results also in significant increases of citrate synthase and B-hydroxyacyl-CoA dehydrogenase, which enzymes are important in the aerobic metabolic system. (S. Mason & Johnson, 2007)
Exercise induces increased HIF-1α and increased HIF-2α mRNA levels, after endurance exercise training these levels are not increased by exercise (Lundby et al., 2006). It is unknown if these regulation of HIF mRNA only is induced by exercise without cellular hypoxia. If that is true, HIF would play a role in the early adaptive response for exercise. The increased HIF mRNA levels did occur after exercise in the untrained leg, while not in the trained leg. The levels increased during the recovery. It is plausible that six hours after exercise there is no hypoxia due to exercise in the muscular cells. The measurements and assumptions indicate that HIF plays a role in the early adaptive response to exercise. Lundby et al. conducted an experiment with trained and untrained legs, within one person. Their goal was to test if a single prolonged exercise bout increases HIF-1α and/or HIF-2α mRNA levels in the untrained leg, while the expression of both HIFs reduces in the trained leg. The subjects trained one leg and the measurements in both legs were compared. In the untrained leg exercise induces increased HIF-1α and increased HIF-2α mRNA levels. In the trained leg no increased mRNA levels were found. They concluded that a single exercise bout in an untrained leg induces increased level of HIF-1α mRNA and HIF-2α mRNA, whereas these increase did not occur in trained legs. This shows that the mRNA expression of HIF-1α and HIF-2α increases with acute exercise and implies that an upregulation of HIF-1α and HIF-2α can be important for the regulation of de early adaptive response to endurance exercise. (Lundby et al., 2006) As Lundby et al. mentioned it is an important question if the upregulation of the mRNA levels of HIF-1α and HIF-2α are induced by cellular hypoxia. According the experiment of Lundby et al. it is unlikely that the HIF-a mRNA induction is dependent of cellular hypoxia, because the induction occurs after 6 hours, while cellular hypoxia should not exist after 6 hours. Secondly are the HIF-1α and HIF-2α mRNA levels higher in the trained leg at rest, while it is unlikely that there are differences in oxygenation levels at rest between trained and untrained muscle. According to Lundby et al. the explanation of the inhibition of HIF-a induction could be explained by an alternative mechanism.
1.2 Cellular hypoxia caused by endurance exercise
To compare endurance exercise with exposure to high altitude, it is necessary to find a common factor. In this report we test if cellular hypoxia could be the common factor which induces muscular adaptations. This paragraph focusses on the question if cellular hypoxia does occur during endurance exercise. First some studies will be discussed, which found reduced cellular oxygen levels. Secondly an explanation for these reduced oxygen levels will be given.
During exercise, the cells in the skeletal muscle tissue use oxygen. This oxygen is used to provide energy. The capillary PO2 in the skeletal muscle reduces during exercise and when the intensity of the exercise increases (Richardson, Noyszewski, Kendrick, Leigh, & Wagner, 1995). Richardson et al. measured the capillary PO2 in six healthy competitive athletes during exercise. These reduced oxygen levels can be explained by increased oxygen consumption.
Oxygen is used for metabolic pathways, in aerobic cellular energy metabolism oxygen is used and carbon dioxide is formed. Glucose is an important energy substrate during exercise. Its catabolism involves 3 subsequent steps. Firstly glycolysis, which converts glucose into pyruvate generating a release of energy. Secondly, the citric acid cycle, which produces ATP, high-energy electrons (NADH, FADH2) and carbon dioxide. And thirdly the electron transport system (ETS), which consists of mitochondrial proteins, located in the inner mitochondrial membrane. The synthesis of ATP in the ETS is also called oxidative phosphorylation. This system requires oxygen as the final electron donor and H+-acceptor. Glycolysis provides ATP without oxygen consumption. The other two processes only occur with enough oxygen and provide more ATP. In case of insufficient oxygen levels, the citric acid cycle and oxidative phosphorylation cannot occur. In such a case, pyruvate is converted to lactate to reduce NADH to NAD+. (Silverthorn, 2013)
Summarizing we can conclude that endurance exercise improves the oxidative capacity in the muscle cells by increased mitochondrial density, increased mitochondrial enzyme activity and a more efficient energy coupling. Endurance exercise leads to inhibition of ROS production and indicates an adaptation of the energy metabolism, this is also supported by Rösler et al., who measured that lactate levels of 4 mM were reached at a higher output. Endurance exercise inhibits also the induction of HIF-a mRNA level, what leads to a glycolytic energy metabolism and less O2-consumption. Furthermore, it seems logical that the cellular O2 levels in skeletal muscular cells reduce, because the oxygen consumption increases. Unfortunately there is no strong evidence to conclude that cellular hypoxia does occur. Richardson et al. measured reduced capillary PO2 levels when the exercise intensity increased. This reduction can be explained by an increased oxygen consumption. If these reduced oxygen levels are responsible for the muscular adaptations in response to exercise, is unclear. In the second chapter we will conduct if these adaptations are caused by reduced oxygen levels.
2. Hypothesized mechanisms mediating adaptation to endurance exercise
The previous chapter discussed the various muscular adaptations in response to endurance exercise. The important question how these adaptations occur is still unanswered. Endurance exercise causes reduced oxygen levels and increased levels of HIF-1α, PDK-1 and reactive oxygen species (ROS). In this chapter we discover how these measurements can be related. In literature two pathways are found, where P53 and HIF-1α are two important factors. Both of them are regulated by cellular oxygen levels and lead to increased oxidative capacity and increased oxygen consumption. ROS results from increased oxygen consumption. In this chapter first the tumour protein p53 will be discussed. Secondly the pathway of HIF-1α will be declared and thirdly something will be said about ROS.
2.1 Tumour suppressor protein P53 and endurance exercise
P53 is a necessary protein in the adaptive changes in metabolism and contributes to mitochondrial biogenesis (production of mitochondria) in response to endurance exercise. (Bartlett, Close, Drust, & Morton, 2014; Park et al., 2009) Tachtsis et al. measured the amount of p53 protein in the nucleus, mitochondria and cytoplasm in muscle biopsies before and after exercise compared with an control group that rested instead of exercised. They found an increase in nuclear p53 in skeletal muscle (m. vastus lateralis) after 3 hours endurance exercise, whereas no changes in cytoplasmic or mitochondrial p53 expression was found. (Tachtsis, Smiles, Lane, Hawley, & Camera, 2016) These results show that the response of p53 to endurance exercise is located in the nucleus.
P53 is necessary for the adaptive changes in aerobic metabolism to increase the exercise capacity in response to training. Park et al. concluded this after evaluating the maximum exercise capacity in p53+/+ and p53-/- mice after a 5-week period of treadmill training. The maximum work capacity and peak VO2 were significantly increased in p53+/+ mice, while the p53-/- mice were relative unresponsive to training. The blood lactate levels stay constant after training in p53+/+ mice, while in p53-/- mice blood lactate levels increased 6-fold after training. P53+/+ have a higher mitochondrial DNA (mtDNA) content in skeletal muscle groups than p53-/- mice. This difference was bigger in aerobic muscle compared to glycolytic muscles, that suggest that the adaptive changes for aerobic metabolism may require p53. (Park et al., 2009)
P53 induces mitochondrial biogenesis by interaction with and regulation of Mitochondrial Transcription Factor A (TFAM) expression. TFAM is an important gene for transcription and maintenance for mtDNA and regulates mtDNA. Park et al. examined the effect on swimming endurance of 3 different genotypes of mice: wild-type (p53+/+), heterozygous (p53+/-) and homozygous (p53-/-) knockout animals. After exercise the TFAM transcript levels were higher in wild-type mice relative to mutant p53 in untrained mice, TFAM mRNA was expressed and peaked 12h after exercise. Because the relative increase in p53+/+ and p53-/- mice were comparable, other factors are probably also important by initiating changes in TFAM expression. Although p53 increases the expression of TFAM. (Park et al., 2009)
Matoba et al. concluded that the inactivation of p53 decreases the dependence on oxygen, which promotes growth in hypoxic environments (Matoba et al., 2006) This is consistent with the data that p53 induces mitochondrial biogenesis. With induction of mitochondrial biogenesis, the cells uses more oxygen and become more dependent on oxygen. Inactivation of p53 logically leads to decreased dependence on oxygen, for mitochondrial biogenesis is inhibited.
2.2 HIF-1α and muscular adaptation
Secondly we will look at HIF-1α’s role in the muscular adaptation. As mentioned before are under hypoxic conditions higher concentrations of HIF-1α found. Even during exercise reduced higher levels of HIF-1α are measured (Lindholm & Rundqvist, 2016). In this paragraph the effects of activated HIF-1α will be mentioned and the mechanism of muscular adaptation induced by HIF-1α will be discussed.
Reduced levels of HIF-1 seems to improve the oxidative capacity and endurance performance (Lindholm & Rundqvist, 2016), what is supported by Mason et al. in their study to the effect of HIF-1α in mice. The found in mice with a skeletal muscle-specific deletion of HIF-1α a better endurance performance and several features associated with a trained muscle. (S. Mason & Johnson, 2007) HIF-1 decreases the oxygen consumption by regulating the mitochondrial function trough increased expression of PDK-1 (Kim et al., 2006), which inhibits the pyruvate dehydrogenase complex (PDHc) and provides an active repression of mitochondrial function (Lindholm & Rundqvist, 2016).
Mason et al. created mice specifically lacking skeletal muscle HIF-1α and subjected them to an endurance training protocol to examine the role of HIF-1α in endurance training. They found that only wildtype mice improve their oxidative capacity, while the null mice had already upregulated this parameter without training. Furthermore, the untrained null mice had an increased capillary to fibre ratio and elevated oxidative enzyme activities what means that both oxygen delivery and oxygen consumption are increased. Additionally, the HIF-1α null muscles have decreased expression of pyruvate dehydrogenase kinase I (PDK-I), this corresponds with the fact that PDK-1 is a target of HIF-1α. These data demonstrate that removal of HIF-1α causes an adaptive response in skeletal muscle skin to endurance training and provides evidence for the suppression of mitochondrial biogenesis by HIF-1α in normal tissue. Based on the values for respiratory exchange ratio (RER, CO2-production/O2-consumption) in WT mice during the endurance test, which was higher than the RER in trained WT. A lower RER indicates a higher O2-consumption, what means a more oxidative metabolism. This indicates that WT mice indeed were able to shift toward a more oxidative metabolic profile. This shift was absent in the trained HIF-1α null mice: their RER values were nearly identical to their untrained values, although they had already lowered RER values, compared to WT mice. This finding indicates that loss of HIF-1α gives rise to a phenotype mimicking exercise training. The HIF-1α null mice have undergone an adaptive process, leading to being better suited for endurance training; endurance training appeared to be unable to further improve upon this adaptation. (S. D. Mason et al., 2007)
Both HIF-1α and p53 responds to endurance exercise and induces gene expression, however HIF-1α and p53 have different effects. P53 increases in the skeletal muscular nucleus after endurance exercise and is necessary for the adaptive changes in aerobic metabolism. P53 induces a shift from aerobic metabolism to more glycolytic metabolism, what is found in higher amounts of mtDNA in especially glycolytic muscles. The mtDNA is regulated by TFAM, which expression is induced by P53. HIF-1α induces glycolysis, glucose uptake and angiogenesis, what results in increased oxygen delivery and relative decreased oxygen consumption, since glycolysis is an anaerobe pathway to produce ATP. HIF-1 increases the expression of PDK-1, which inhibits the pyruvate dehydrogenase complex (PDHc). The inhibition of PDHc leads to reduced mitochondrial function, what results in an decreased oxygen consumption.
3. The effect of high altitude
In the previous chapter we have seen that exercise in reduced oxygen levels and therefore induces muscular adaptations to increase the oxidative capacity. However, oxidative stress is not only caused by exercise, but can also be observed at altitude without strenuous physical exertion (Askew, 2002). In this chapter we will have a look at the muscular adaptation to exposure to high altitude and the cellular hypoxia that is assumed to be at high altitude. The first paragraph focusses on cellular hypoxia and the second paragraph will handle the muscular adaptations to high altitude.
3.1 Cellular hypoxia caused by high altitude
Oxygen is continuously used to provide energy through oxidative phosphorylation. Oxygen is available in the air and is inhaled through the lungs, where it enters the blood by diffusion, after which it eventually arrives in the cells via the bloodstream. The oxygen concentration in the pulmonary vein depends on the air pressure and oxygen concentration.
The air pressure lowers when altitude increases, which will result in lower partial oxygen pressure (PO2)) levels in the blood. The percentage of oxygen in air is constant at different altitudes, but the atmospheric pressure decreases at higher altitude. This lowered pressure decreases also the partial pressure of inspired oxygen and with it the PO2 in the arterial blood. (Flaherty et al., 2016; Peacock, 1998) This lowered PO2 causes a reduced haemoglobin-oxygen saturation and reduced total oxygen content of the blood. This stressor initiates important physiological adaptations to compensate for this hypoxic stress. (Flaherty et al., 2016) These adaptations will be mentioned in the next paragraph. The lowered oxygen concentration at high altitude is called hypobaric hypoxia.
Richardson et al. measured the capillary PO2 during normoxic and hypoxic conditions and found a reduced mean capillary PO2 during hypoxia (Richardson et al., 1995). Although it is measured that hypoxia causes capillary hypoxia, there were no experiments found where the cellular PO2 is measured.
3.2 Muscle adaptations to high altitude
In this paragraph the muscular adaptations to high altitude will be pointed out. After these adaptations are listed even some possible mechanisms will be described. Three major adaptations which are found are metabolism switch, change in oxygen consumption and a more discussed adaptation is de change in mitochondrial density.
The Pasteur effect is an important reaction to hypoxia. The Pasteur effect is an metabolic reaction and means that the oxidative phosphorylation reduces and the anaerobe fermentation increases. The glycolytic pathway increases to maintain the cellular ATP levels, because oxidative phosphorylation produces less ATP per glucose molecule. In mammalian cells, this metabolic switch is regulated by HIF-1. (Seagroves et al., 2001)
With this metabolism switch also the oxygen consumption changes, i.e., oxygen consumption is reduced (Levett et al., 2012). After 6 weeks of intermittent hypoxic training in athletes, elevated GLUT-4 transport mRNA has been recorded in skeletal muscle (Flueck, 2009), which facilitates a longer-lasting glucose uptake. These longer-lasting glucose uptake is important for longer energy supply in the muscular cells. In this way we can conclude that hypoxic exercise leads to a switch in metabolism, as Kim et al. mentioned in their paper.
Levett et al conducted an acclimatization study to the skeletal muscle mitochondria at high altitude hypoxia. No significant changes in mitochondrial density were found, after 19 days exposure to high altitude hypoxia. (Levett et al., 2012) This result does not correspond with the results of the study of Jacobs et al. They studied the effects of high altitude (3454 m) with a maintained energetic balance. The subjects tried to have the same physical activity level and the same diet. They found that the skeletal muscle mitochondrial volume density increased, with specific increased intermyofibrillar mitochondrial volume density and constant subsarcolemmal mitochondrial volume density. (Jacobs et al., 2015) The explanation for the difference can be found in the difference of energetic balance. Jacobs et al. maintained the energetic balance, were Levett et al. conclude that loss of body mass is something that occurs on altitude exposure due to loss of efficiency. Besides that, both experiments used only few subjects (6 subjects in the experiment of Levett and 9 in Jacobs’ experiment) which makes it hard to have significant results. Edwards et al studied also the response to high altitude hypoxia. They studied the skeletal muscle function and concluded that the skeletal muscle function is maintained, although significant atrophy was measured. (Edwards et al., 2010)
Levett also measured an trend of UCP3 mRNA and protein upregulation, which seems to protect mitochondria from excessive ROS. The mitochondrial density and citrate synthase seems to reduce, with down-regulation of PGC1α. These adaptations suggest a coordinated response to downregulate the mitochondrial biogenesis to correspond the oxygen availability with the oxygen consumption. This is in contradiction with the response to exercise, where mitochondrial biogenesis is induced.
Notably, Levett et al. did not found increased HIF-1α during the altitude experiments, this can be explained with a possible degradation of HIF-1 during sampling of the biopsies. The PPAR-α expression is increased, what can result in a switch to fatty acid oxidation, Levett et al. found non-significant loss of intramyocellular fat stores, which supports the capacity to use fat as substrate. (Levett et al., 2012)
High altitude reduces the partial oxygen pressure in blood levels, what causes reduced haemoglobin-oxygen saturation and reduced total oxygen content of blood. To compensate for this hypoxic stress, physiological adaptations are initiated. There are no studies found that measured the cellular oxygen levels. So we cannot say that at high altitude cellular hypoxia occurs, but we know that in the muscular capillary P02 is also reduced. To compensate for hypoxic stress, the oxidative phosphorylation reduces and the anaerobe fermentation increases. The glycolytic pathway increases to maintain the cellular ATP levels. This switch is regulated by HIF-1. With this switch, the oxygen consumption reduces. If the mitochondrial density changes remains controversial, although it is clear that the relative subsarcolemmal mitochondrial density is reduced.
4. Effects high altitude training: exercise at high altitude
After we studied the muscular adaptations to endurance exercise and to high altitude exposure, we will have a look at the effects of high altitude training, which means that exercise and high altitude are combined. In this chapter different studies will be discussed and the contribution of cellular hypoxia to the muscular adaptations will be discussed.
The results of studies from Desplanches et al. suggest that the increased muscle capillarity and mitochondrial volume density are caused by exercise, whereas the increased mean fibre cross-sectional area is initiated by hypoxia. Desplanches et al. compared muscle structural adaptations induced by an exercise program combining hypoxia and severe exercise training to those occurring with training at the same relative workload in normoxia. There was a control group training in normoxia (Con T), a second group training under hypoxic conditions (Hyp T), the subjects of the Hyp T group performed a second training period with the same absolute workload, but in normoxia (Nor T). Muscle biopsies were taken before and after 3 weeks of training consisting of cycle ergometer exercise in sever progressive normobaric hypoxia (equivalent to altitudes 4100 – 5700 m) or in normoxia. Con T & Hyp T had the same effects on muscle capillarity and mitochondrial volume density, both of them were increased. The increased mitochondrial volume density is found in both the control group and the group training under hypoxic conditions. These increased mitochondrial volume densities are probably results of the exercise training, because there are no significant difference between the Con T and the Hyp T group.(Desplanches et al., 1993) This supports the results from chapter 1 were we found that endurance exercise increases the mitochondrial volume density. Under hypoxic conditions, the mean fibre cross-sectional area increased also. That supports that local hypoxia induces adaptation in endurance exercise.
Green et al. studied the effect of exercise to the muscle metabolism during acute (4 hours) and chronic (3 weeks) hypoxia (similar to altitude of 4300 m). Exercise performed during acute hypoxia resulted in a greater increase compared to rest in muscle lactate concentration than exercise performed either at sea level or during chronic hypoxia. (Green et al., 1992) The smaller increase of muscle lactate concentration suggest that the muscle metabolism could adapt to prolonged hypoxia. The ATP concentrations in the muscle did not change after prolonged exercise. These ATP concentrations are also maintained with acclimatization to high altitude. So the muscle ATP homeostasis seemed to be maintained during prolonged whole body exercise of moderate intensity after acute hypoxia. Because the fibre size and cappilarization in the muscle are not changed, the ATP homeostasis is apparently not depend from changes in mitochondrial capillarity, fibre size or cappilarization. The ATP homeostasis seems to be maintained by reduction of the lactate production, what leads to more efficient coupling between oxidative phosphorylation and glycolytic flux. (Green et al., 1992)
In studies were hypoxia and exercise were combined no additive effects were found. We have seen that the effects accumulated and did not have opposite effects. However, it is hard to conclude from these studies that the effects of endurance exercise and hypoxia are both induced by cellular hypoxia. However Desplanches et al. concluded that the muscle tissue adaptation in endurance exercise is induced by local hypoxia, rather than metabolic flux rate.
Discussion and Conclusion
This study started with the question if the skeletal muscular adaptations in response to endurance exercise are caused by cellular hypoxia. The hypothesis that the adaptation of muscle metabolism due to exercise training is caused by cellular hypoxia in the muscle tissue, was tested by comparing the effects of exposure to high altitude and the effects of exercise training.
Endurance exercise appeared to improve the oxidative capacity by increased mitochondrial density, increased mitochondrial enzyme activities and efficient energy coupling. HIF-1α and p53 appeared as proteins which induces these effects. Both proteins induces the energy metabolism shift to anaerobic and HIF-1α increased the oxygen delivery, by inducing angiogenesis. P53 induces the mitochondrial biogenesis what results in increased oxidative capacity. P53 in the nucleus of skeletal muscular cells increases the expression of TFAM, what induces mitochondrial biogenesis. HIF-1 increases PDK-1, which inhibits the pyruvate dehydrogenase complex wat leads to reduced mitochondrial function an reduces the oxygen consumption. There are no studies found which related p53 or PDK-1 to the effects of high altitude. This suggest different patterns between adaptions to endurance exercise and adaptations to high altitude.
During high altitude oxygen consumption is reduced and the energy metabolism switches from aerobic to anaerobic. HIF-1 is found during high altitude and is suggested to play a role in this switch. Because endurance exercise and high altitude exposure have corresponding effects on the skeletal muscular tissue, it is plausible to assume that these corresponding effects are initiated by HIF-1. Nevertheless there are just a few studies which propose HIF-1 as factor to induce muscular adaptations in response to high altitude. Different studies show that HIF-1 reduces the oxygen consumption by the reduction of the mitochondrial function. HIF-1 increases PDK-1, which inhibits the pyruvate hydrogenase complex whereby the mitochondrial function is reduced. If the upregulation of HIF-1 is induced by cellular hypoxia is dubious, although lowered oxygen concentrations in blood and upregulated HIF-1α mRNA levels are shown both during exercise and during high altitude.
High altitude and endurance exercise have shown different effect on mitochondrial biogenesis. Where exercise increases mitochondrial biogenesis, high altitude reduces mitochondrial biogenesis (Levett et al., 2012). This finding does not support the hypothesis that the effects of high altitude and endurance exercise are both induced by cellular hypoxia.
The high altitude training studies did not show accumulated effect of endurance training were during exposure to high altitude. These findings do not correspond with the hypothesis that the muscular adaptations to endurance exercise are caused by the same stressor compared to muscular adaptations to high altitude.
After all, we cannot conclude that the skeletal muscular effects of endurance exercise are caused by cellular hypoxia. Further research need to be done, especially to the cellular oxygen levels during exercise and high altitude. This can be done with a study where cellular hypoxia in the skeletal muscle tissue is imitated and the effects are measured. This can be done with mice in chambers with lowered oxygen concentrations, where the capillary oxygen levels and the cellular oxygen levels are measured. The cellular oxygen pressure can be measured while the mice are in the hypoxic chambers. The cellular oxygen concentrations can possibly be measured with MRS. Otherwise can a muscle biopsies be taken before, during and after exposure to hypoxia, which can be evaluated for their oxygen concentrations. When these effects correspond to the effects of endurance exercise, it is more reliable that endurance exercise and cellular hypoxia have a causal relation. The last study object then is to conduct the mechanism by which cellular hypoxia leads to skeletal muscular adaptations.